Novel triazine-based detoxification agents and their use

ABSTRACT

An affinity ligand-matrix conjugate comprises the matrix and conjugated thereto by the group Z, a ligand having general formula (I):  
                 
 
wherein one X is N and the other X is N, CCL or CCn; A 1  and A 2  are each independently O, S or N—R 1  and R 1  is H, C 1-6  alkyl, C 1-6  hydroxyalkyl, benzyl or β-phenylethyl; B 1  and B 2  are each independently an optionally substituted hydrocarbon linkage containing from 1 to 10 carbon atoms; D 1  is H or a primary amino, secondary amino, tertiary amino, quaternary ammonium, imidazole, guanidine or amidino group; and D 2  is a secondary amino, tertiary amino, quaternary ammonium, imadazole, guanidine or amidino group; or B 2 -D 2  is —CHCOOH—(CH 2 ) 3-4 —NH 2 ; and p is  0  or  1 . Such conjugates are useful for the separation, isolation, purification, characterization, identification or quantification of an endotoxin.

FIELD OF THE INVENTION

The present invention relates to novel affinity ligands, theirpreparation and attachment to matrices which may consist of solid,semi-solid, particulate or colloidal materials, or soluble polymers. Theinvention furthermore relates to these novel affinity ligand-matrixconjugates and the preparation and use thereof in the binding andremoval of endotoxin from various fluids such as water, aqueoussolutions, body fluids, blood, plasma, solutions of pharmaceuticalproducts, proteins and other compounds of biological origin.

BACKGROUND OF THE INVENTION

Endotoxins are lipopolysaccharides found in the outermost membrane ofGram-negative bacteria, particularly pathogeneic bacteria of the classEnterobacteriaceae, Neisseriaceae and Chlamydiaceae. Endotoxins compriselipid A attached to a polysaccharide of variable structure dependentupon its biological origin. The polysaccharide component ofEnterobacteriaceae endotoxin is characterised by an O-specific chainregion and a core region. The O-specific region comprises up to 50repeating oligosaccharide units that contain as many as 8 differentsugar residues. O-specific chains exhibit large structural diversityfrom species to species whereas the core region, divided into the outercore and inner core regions, is less variable. The inner core region ischaracterised by the presence of unusual sugar residues such as heptoseand 2-keto-3-deoxyoctonic acid (KDO) which are frequently substitutedwith phosphate or phosphate derivatives. Also attached to the inner coreregion, lipid A is a conserved biphosphorylated glucosamine disaccharidewhich is acylated by 4 saturated primary acyl groups of which 2 carrysecondary saturated acyl groups. The combination of hydrophobic lipid Atails with the hydrophilic and anionic polysaccharide unit providesendotoxin with amphipathic properties.

Endotoxin released from the cell wall of Gram-negative bacteria isconsidered to be the primary cause of the many pathiphysiologicaloccurrences that accompany Gram-negative septicaemia. Endotoxin at pg/mlconcentrations in blood triggers the release of a variety of cytokines,including interleukins and TNF. Over stimulation of the immune system byendotoxin leads to a massive release of cytokines which ultimatelyresults in metabolic breakdown and septic shock. During septic shock,the complement and coagulation cascades become activated and vascularpermeability increases. This can lead to disseminated intravascularcoagulation and multiple organ failure, often with fatal consequences.Septic shock often develops because of the lack of an initial responseto infection allowing the level of blood-borne endotoxin to reachcritical levels.

In addition to the obvious risk presented by the presence of live Gramnegative bacteria or cell wall debris in parenteral pharmaceuticalproducts, the presence of free endotoxin in pharmaceutical preparationsis also a major concern. Because endotoxin is such a potent immunestimulator, very low concentrations may cause toxic reactions includingpyrogenic effects. Endotoxin is a relatively stable molecule which isnot inactivated by routine autoclaving or treatment with organicsolvents. Exposure to concentrated sodium hydroxide or prolonged hightemperature (250° C.) will inactivate endotoxin, though such methods arenot appropriate for most biological products. Furthermore, maintenanceof complete sterility throughout the manufacture of bio-therapeutics isproblematic. Consequently, the highly efficient capture and removal ofendotoxin from parenteral pharmaceuticals is very desirable,particularly in situations where endotoxin is known to associate withcomponents of the therapeutic formulation.

A variety of techniques have been used to remove endotoxin from aqueoussolutions including ultrafiltration, charcoal adsorption,cation-exchange chromatography, and a variety of immobilised affinityligands including polymyxin B and endotoxin binding protein. All ofthese techniques exhibit significant shortcomings, particularly in thecase of endotoxin removal from high molecular weight compounds such astherapeutic proteins. Ultrafiltration can only be used to removeendotoxin from low molecular weight compounds whereas charcoaladsorption tends to promote the binding of most organic compounds.Cation-exchange chromatography is effective in removing endotoxin fromwater but less effective for protein containing solutions, particularlyproteins with acidic isoelectric points. Polymyxin B, a cyclicpolypeptide antibiotic, is too toxic to allow its use for thepurification of therapeutic products whereas endotoxin binding proteinis too expensive for commercial applications.

Immobilised cationic amino acids (histidine, lysine and arginine) havealso been used for endotoxin removal (Tosa, T. et al., MolecularInteractions in Bioseparations, Ed. Ngo, T. T., Plenum Press, New York,pp. 323-332, 1993; Lawden, K. H. et al., Bacterial Endotoxins:Lipopolysaccharides From Genes to Therapy, Wiley-Liss Inc., pp. 443-452,1995). Such materials have been prepared by direct attachment of aminoacids to epoxy-activated chromatographic matrices. In the case ofPyrosep™, a commercially available lo material manufactured by TanabeSeiyaku Company Limited, Osaka, Japan, a single histidine group isimmobilised to a support matrix by a hexanediamine spacer arm. Again,such materials are adequate for removal of endotoxin from water orsolutions of low molecular weight compounds, but their performance iscompromised in the presence of salt (>50 mM) or proteins which have anaffinity for endotoxin. Consequently, none of the existing methods ofendotoxin removal are suited to the elimination of endotoxin frombio-therapeutic compounds intended for parenteral administration. Thisis especially true for protein therapeutics where no single effectiveand safe method of endotoxin removal exists.

Removal of endotoxin from blood or plasma may provide an effectiveapproach to the management of septic shock, particularly if applied atthe early stages of infection or prophylactically in situations where anincreased risk of septic shock is anticipated (e.g. major bowel or liversurgery). Several studies have been reported as to the use ofmonocolonal antibodies directed against endotoxin or cytokines releasedin the initial phase of the shock reaction. However, most of theseapproaches have been found to be ineffective (Siegel, J. P., DrugInformation Journal, 30, pp. 567-572, 1996). In contrast, extracorporealextraction of endotoxin from whole blood has been accomplished by use offibre-immobilised polymyxin B (Aoki, H. et al., Nippon Geka GakkaiZasshi (Japan), 94, pp. 775-780, 1993), though concerns over potentialtoxicity of polymyxin B lechates remain. Consequently, affinityadsorbents incorporating endotoxin binding ligands which have highaffinity for endotoxin and low toxicity may also be beneficial for themanagement of sepsis.

Immobilised amino acids have also been investigated as potentialendotoxin removal agents but such materials bind endotoxin weakly andnon-specifically and are of limited value in the extraction of endotoxinfrom biological fluids and solutions of biological compounds.Triazine-based compounds have been reported which bind selectively toproteins; however, such ligands are not applicable to the isolation ofendotoxin.

SUMMARY OF THE INVENTION

This invention relates to the discovery of synthetic affinity ligandstructures which bind selectively to endotoxin. A generic group of novelaffinity ligands have been found which exhibit high affinity forendotoxin and are generally applicable to the isolation of endotoxinfrom a variety of sources.

A feature of the present invention is the provision of a general toolfor the removal of endotoxin contamination from biological materials.Endotoxin binds exceedingly tightly to affinity ligand-matrix conjugatesof the invention. This feature enables highly efficient extraction ofendotoxin from water and aqueous solutions providing a means ofgenerating pyrogen-free water or pyrogen-free solutions. Affinityligand-matrix conjugates of the invention are especially valuable forthe removal of endotoxin which is bound to or associated with proteins,drugs or other biological compounds intended for medical orpharmaceutical applications. Certain biological compounds, particularlyproteins, often bind endotoxin tightly and subsequent removal is verydifficult, if not impossible, by existing means. Affinity ligand-matrixconjugates of the invention may also be applied to the removal ofendotoxin from blood or plasma and so provide an especially useful toolfor in vitro or in vivo removal of endotoxin, the latter being achieved,for example, by way of an extracorporeal endotoxin extraction device.Such a device may be especially valuable for removal of endotoxin whichis released into the blood stream during bacterial infections, suchinfections often causing life-threatening diseases such as septicaemiaor meningitis. Removal of blood-borne endotoxin may be particularlybeneficial in the treatment of these diseases and in the prevention andmanagement of septic shock.

Novel affinity ligand-matrix conjugates provided by this invention canbe used in place of other endotoxin binding materials and aresignificantly more flexible in their use, are more robust, lessexpensive to produce and offer greater endotoxin binding efficiencies.

The present invention relates to affinity ligand-matrix conjugatescomprising a ligand having General Formula (1):

wherein one of the symbols X represents a nitrogen atom and the othersymbol X represents a nitrogen atom or a carbon atom carrying a chlorineatom or a cyano group;

-   -   A₁ and A₂ each independently represent an oxygen atom, a sulphur        atom or a group N—R₁;    -   R₁ represents a hydrogen atom, an alkyl group containing from 1        to 6 carbon atoms, a hydroxyalkylgroup containing from 1 to 6        carbon atoms, a benzyl group or a β-phenylethyl group;    -   B₁ and B₂ each independently represent an optionally substituted        hydrocarbon linkage containing from 1 to 10 carbon atoms (any        substituent being substantially non-critical with respect to        utility) and including alkyl, phenyl, naphthyl and cyclohexyl        groups;    -   D₁ represents a hydrogen atom, a primary amino group, a        secondary amino group, a tertiary amino group, a quaternary        ammonium group, an imidazole group, a guanidino group or an        amidino group;    -   D₂ represents a primary amino group (e.g. as derived from lysine        or ornithine), a secondary amino group, a tertiary amino group,        a quaternary ammonium group, an imidazole group, a guanidino        group or an amidino group; and    -   p is 0 or 1.

The ligand is attached to a support matrix in position Z, optionallythrough a spacer arm interposed between the ligand and matrix.Alternatively, in novel ligands of the invention, Z represents afunctional group of the type capable of reaction with a solid matrixthat may be activated (if necessary or desired) or unactivated.

DESCRIPTION OF THE INVENTION

When conjugated to a matrix, the optional spacer arm is preferablyrepresented by General Formula (II):-T-[L-V]_(m)-   (II)wherein T represents an oxygen atom, a sulphur atom or a group N—R₂;wherein R₂ represents a hydrogen atom or an alkyl group containing from1 to 6 carbon atoms;

-   -   V represents an oxygen atom, a sulphur atom, a —COO— group, a        CONH group or an NHCO group, a —PO₃H group, a        NH-arylene-SO₂—CH₂—CH₂-group or a N—R₃ group; wherein R₃        represents a hydrogen atom or an alkyl group containing from 1        to 6 carbon atoms;    -   L represents an optionally substituted hydrocarbon linkage        containing from 2 to 20 carbon atoms; and    -   m is 0 or 1.

The support matrix may be any compound or material, particulate ornon-particulate, soluble or insoluble, porous or non-porous which may beused in conjunction with affinity ligands to form an affinityligand-matrix conjugate and which provides a convenient means ofseparating the affinity ligands from solutes in a contacting solution.

The present invention provides novel affinity ligand-matrix conjugates,which affinity ligand-matrix conjugates may be used in the isolation orremoval of endotoxin from water, aqueous solutions, body fluids, blood,plasma, solutions of pharmaceutical products, proteins and othercompounds of biological origin.

In a preferred embodiment, the invention provides novel affinityligand-matrix conjugates which are represented by the General Formula(III):

wherein A₁, A₂, B₁, B₂, D₁, D₂, p, X, T, L, V, m, R₁, R₂, and R₃ havethe meanings specified above; and M represents the residue of a supportmatrix which may be any compound or material, particulate ornon-particulate, soluble or insoluble, porous or non-porous which may beused in conjunction with affinity ligands to form an affinityligand-matrix conjugate and which provides a convenient means ofseparating the affinity ligands from solutes in a contacting solution.

It will be appreciated that this invention relates, inter alia, to theuse of compounds which are pyrimidines, diazines, or triazines carryinga -T-[L-V]₀₋₁-M substituent, or the precursor thereof, and othersubstituents linked to the ring via a hetero atom. Such substituents mayinclude any non-interfering group comprising 0 to 20 carbon atoms.

In the present specification, whenever the term endotoxin is used in aplural or generic sense, it is intended to mean endotoxins originatingfrom any microbiological source. By the term endotoxin is thus alsomeant lipopolysaccharide from any species including Enterobacteriaceae,Neisseriaceae and Chlamydiaceae. Since endotoxin is known to beheterogeneous, the term “endotoxin” as used herein includes allnaturally occurring forms which comprise lipid A covalently linked to apolysaccharide, including analogues, derivatives, fragments andprecursors thereof.

The term “primary amino group” as used herein, alone or in combination,refers to an —NH₂ group.

The term “secondary amino group” as used herein, alone or incombination, refers to a —NHR₄ group; wherein R₄ represents a straightor branched alkyl group containing from 1 to 6 carbon atoms.

The term “tertiary amino group” as used herein, alone or in combination,refers to a —NR₅R₆ group; wherein R₅ and R₆ each represent a straight orbranched alkyl group containing from 1 to 6 carbon atoms.

The term “quaternary ammonium group” as used herein, alone or incombination, refers to a —NR₇, R₈, R₉+ group; wherein R₇, R₈ and R₉ eachrepresenta straight or branched alkyl group containing from 1 to 6carbon atoms.

The term “alkyl group containing from 1 to 6 carbon atoms” as usedherein, alone or in combination, refers to a straight or branched,saturated hydrocarbon chain having 1 to 6 carbon atoms such as e.g.methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, n-hexyl,4-methylpentyl, neopentyl and 2,2-dimethylpropyl.

The term “hydroxyalkyl group containing from 1 to 6 carbon atoms” asused herein, alone or in combination, refers to a straight or branched,saturated hydrocarbon chain having 1 to 6 carbon atoms substituted withone or more hydroxy groups, preferably one hydroxy group, such as e.g.hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl,4-hydroxybutyl, 5-hydroxypentyl and 6-hydroxyhexyl.

The term “alkoxy group containing from 1 to 6 carbon atoms” as usedherein, alone or in combination, refers to a straight or branchedmonovalent substituent comprising an alkyl group containing from 1 to 6carbon atoms linked through an ether oxygen having its free valence bondfrom the ether oxygen and having 1 to 6 carbon atoms e.g. methoxy,ethoxy, propoxy, isopropoxy, butoxy and pentoxy.

The term “halogen” means fluorine, chlorine, bromine or iodine.

The term “acyloxy or acylamino containing from 1 to 6 carbon atoms” asused herein refers to a monovalent substituent comprising an alkyl groupcontaining from 1 to 5 carbon atoms linked through a carbonyloxy oroxycarbonyl group such as a methylcarbonyloxy, ethylcarbonyloxy,methyloxycarbonyl or ethyloxycarbonyl group or linked through acarbonylamino or aminocarbonyl group such as a methylcarbonylamino,ethylcarbonylamino, methylaminocarbonyl or ethylaminocrbonyl group.

The term “alkysulfonyl containing from 1 to 6 carbon atoms” as usedherein refers to a monovalent substituent comprising an alkyl groupcontaining from 1 to 6 carbon atoms linked through a sulfonyl group suchas e.g. methylsulfonyl, ethylsulfonyl, n-propylsulfonyl,isopropylsulfonyl, n-butylsulfonyl, sec-butylsulfonyl, isobutylsulfonyltert-butylsulfonyl, n-pentylsulfonyl, 2-methylbutylsulfonyl,3-methylbutylsulfonyl, n-hexylsulfonyl, 4-methylpentysulfonyl,neopentylsulfonyl, and 2,2-dimethylpropylsulfonyl.

The term “one or more substituents independently selected from” shallmore preferably refer to from 1-3 substituents. The term shall furtherpreferably refer to 1-2 substituents and most preferably refer to onesubstituent.

The term “optionally substituted hydrocarbon linkage containing from 2to 20 carbon atoms” as used herein refers to one or more linear orbranched alkyl chains, optionally substituted with for example hydroxyor alkoxy groups containing from 1 to 6 carbon atoms, and optionallylinked together by amino, ether, thioether, ester, amide or sulphonamidebonds providing a chain containing from 2 to 20 carbon atoms. Theconstruction is preferably flexible. The construction of such optionallysubstituted hydrocarbon linkages is for example described in Lowe, C. R.and Dean, P. D. G, 1974, Affinity Chromatography, John Wiley & Sons,London, which is hereby incorporated by reference.

The term “optionally substituted hydrocarbon linkage containing from 1to 10 carbon atoms” as used herein, alone or in combination, refers to alinear or branched hydrocarbon chain having 1 to 10 carbon atomsoptionally substituted with one or more functional groups, including butnot limited to, carboxyl groups, preferably one carboxyl group, andhydroxyl groups.

In a preferred embodiment of the invention, R₁ represents a hydrogenatom.

In another preferred embodiment of the invention, R₂ represents ahydrogen atom.

In another preferred embodiment of the invention, R₃ represents ahydrogen atom.

In another preferred embodiment of the invention, R₄ represents a methylgroup, an ethyl group or a propyl group.

In another preferred embodiment of the invention, R₅ represents a methylgroup, an ethyl group or a propyl group.

In another preferred embodiment of the invention, R₆ represents a methylgroup, an ethyl group or a propyl group.

In another preferred embodiment of the invention, R₇ represents a methylgroup, an ethyl group or a propyl group.

In another preferred embodiment of the invention, R₈ represents a methylgroup, an ethyl group or a propyl group.

In another preferred embodiment of the invention, R₉ represents a methylgroup, an ethyl group or a propyl group.

In another preferred embodiment of the invention, A₁ represents N—R₁wherein R₁ is as defined above.

In another preferred embodiment of the invention, A₂ represents N—R₁wherein R₁ is as defined above.

In another preferred embodiment of the invention, B₁ represents a—CHCOOH—CH₂— group, a —CHCOOH—(CH₂)₂— group, a —CHCOOH—(CH₂)₃— group, a—CHCOOH—(CH₂)₄— group, an ethyl group, a propyl group, a 2-hydroxypropylgroup, a butyl group, a pentyl group, a hexyl group or a phenyl group.

In another preferred embodiment of the invention, B₂ represents a—CHCOOH—CH₂— group, —CHCOOH—(CH₂)₂— group a —CHCOOH—(CH₂)₃— group, a—CHCOOH—(CH₂)₄— group, ethyl group, a propyl group, a 2-hydroxypropylgroup, a butyl group, a pentyl group, a hexyl group or a phenyl group.

In another preferred embodiment of the invention, D₁ representshydrogen, an amino group, an imidazole group, a guanidino group, anaminidino group, a trimethylammonium group, a triethylammonium group, adimethylamino group, a diethylamino group, a methylamino group or anethylamino group.

In another preferred embodiment of the invention, D₂ represents an aminogroup, an imidazole group, a guanidino group, an aminidino group, atrimethylammonium group, a triethylammonium group, a dimethylaminogroup, a diethylamino group, a methylamino group or an ethylamino group.D₂ (and often also D₁) is preferably a strongly charged species.

In another preferred embodiment of the invention, p represents 0 or 1.

In another preferred embodiment of the invention, both X represent anitrogen atom.

In another preferred embodiment of the invention, T represents an oxygenatom or, more preferably, an NH group.

In another preferred embodiment of the invention, L represents a butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl or dodecyl group and V and mare as defined above.

In another preferred embodiment of the invention, V represents an oxygenatom, a —COO— group, a PO₃H— group or an N—R₃ group; and more preferredan oxygen atom or an NH group and L and m are as defined above.

In another preferred embodiment of the invention, m represents 1 and Land V are as defined above.

The term “integer between x and y” may include the values x (includingzero) and y.

The invention also provides methods for the manufacture of novelaffinity ligand-matrix conjugates according to the invention whichcomprises reacting, in any order,

-   -   (i) a halogenoheterocyclic compound of General Formula (IV):        wherein the symbols X have the meaning hereinbefore specified        and W represents a halogen atom with    -   (ii) a compound of General Formula (V):        D₁-[B₁]_(p)A₁-H   (V)        wherein the symbols D₁, B₁, A₁ and p have the meanings        hereinbefore specified and H is hydrogen,    -   (iii) a compound of General Formula (VI):        D₂-B₂-A₂-H   (VI)        wherein the symbols D₂, B₂ and A₂ have the meanings hereinbefore        specified and H is hydrogen, and    -   (iv) with either an optionally derivatised support matrix of        General Formula (VII):        H-T-]L-V]_(m)-M   (VII)        wherein the symbols T, L, V, m and M have the meanings        hereinbefore specified and H is hydrogen    -   or, with a linking unit of General Formula (VIII):        H-T-L-V-H   (VIII)        wherein the symbols T, L, V have the meanings hereinbefore        specified to give a compound of General Formula (IX):        wherein A₁, A₂, B₁, B₂, D₁, D₂, p, X, T, L, V, m, R₁, R₂, R₃,        R₄, R₅, R₆, R₇, R₈, and R₉ have the meanings hereinbefore        specified; the compound of General Formula (IX) is then reacted        further with a support matrix whose residue is represented by M        using activating and coupling procedures well known to those        skilled in the art.

As examples of halogenoheterocyclic compounds of General Formula (IV)there may be mentioned 5-chloro-2,4,6-trifluoropyrimidine,5-cyano-2,4,6-trichloropyrimidine, cyanuricfluoride, cyanuric bromideand, above all, cyanuric chloride.

As examples of compounds of General Formula (V) there may be mentionedammonia, water, arginine, lysine, histidine, α, γ-diaminobutyric acid,m-aminobenzamidine, p-aminobenzamidine, m-aminobenzenetrimethyl-ammoniumbromide, p-aminobenzenetrimethylammonium bromide,2-(diethylamino)ethylamine, (2-aminoethyl)trimethylammonium chloride,histamine, agmatine, ethylenediamine, 1,3-diaminopropane,1,3-diamino-2-hydroxypropane, 1,4-diaminobutane, 1,5-diaminopentane and1,6-diaminohexane.

As examples of compounds of General Formula (VI) there may be mentionedarginine, lysine, histidine, α,γ-diaminobutyric acid,m-aminobenzamidine, p-aminobenzamidine, m-aminobenzenetrimethylammoniumbromide, p-aminobenzenetrimethylammonium bromide,2-(diethylamino)ethylamine, (2-aminoethyl)trimethylammonium chloride,histamine, agmatine, ethylenediamine, 1,3-diaminopropane,1,3-diamino-2-hydroxypropane, 1,4-diaminobutane, 1,5-diaminopentane,1,6-diaminohexane.

As example of support matrices whose residue is represented by M, theremay be mentioned insoluble support matrices such as a naturallyoccurring polymer, for example a polypeptide or protein such ascross-linked albumin or a polysaccharide such as agarose, alginate,carrageenan, chitin, cellulose, dextran or starch; synthetic polymerssuch as polyacrylamide, polystyrene, polyacrolein, polyvinyl alcohol,polymethylacrylate or perfluorocarbon; inorganic compounds such assilica, glass, kieselguhr, alumina, iron oxide or other metal oxides orco-polymers consisting of any combination of two or more of a naturallyoccurring polymer, synthetic polymer or inorganic compounds. Alsoincluded within the definition of support matrices whose residue isrepresented by M are soluble support matrices comprising polymers suchas dextran, polyethylene glycol, polyvinyl alcohol or hydrolysed starch,which provide affinity-ligand matrix conjugates for use in liquidpartitioning; or support matrices comprising compounds such asperfluorodecalin which provide affinity-ligand matrix conjugates for usein the formation of affinity emulsions. For the avoidance of doubt, asupport matrix is defined herein as any compound or material whetherparticulate or non-particulate, soluble or insoluble, porous ornon-porous which may be used to form a novel affinity ligand-matrixconjugate according to the invention and which provides a convenientmeans of separating the affinity ligand from solutes in a contactingsolution.

Also included within the definition of support matrices whose residue isrepresented by M are support matrices such as agarose, cellulose,dextran, starch, alginate, carrageenan, synthetic polymers, silica,glass and metal oxides which have been, or are, modified by treatmentwith an activating agent prior to, or during, attachment of the ligand.

In a preferred embodiment of the invention, M represents optionallyactivated agarose, silica, cellulose, dextran, glass, toyopearl,hydroxyethylmethacrylate, polyacrylamide, styrenedivinylbenzene, HyperD, perfluorocarbons, polysulphone, polyethersulphone,polyvinylidinefluoride, nylon, and polyvinylchloride. Preferably Mrepresents optionally tresyl-activated, sulphonyl chloride-activated,tosyl-activated, vinylsulphone-activated or epoxy-activated agarose.

There exists a considerable number of activating agents which have founduse for the general purpose of attaching ligands to support matrices.These compounds and their method of use are well known to those skilledin the art and, since the nub of the present invention lies in thenature of the ligand attached to the matrix and not in the mode ofattachment, any of these activating agents will serve in the preparationof the new matrix-ligand conjugates of the invention. As non-limitingexamples of such activating agents there may be mentioned such diversecompounds as cyanogen bromide, cyanuric chlorde, epichlorohydrin, divnylsulphone, p-toluenesulphonyl chloride, 1,1′-carbonyldiimidazole, sodiummeta-periodate, 2-fluro-1-methylpyridiniumtoluene-4-sulphonate,glycidoxypropyltrimethoxysilane and 2,2,2-trifluroethanesulphonylchloride. As indicated above, the procedures by which such activatingsteps are carried out are well known to those skilled in the art.

Similarly, a wide variety of condensing agents may be used to attach thecompounds of General Formulae (VIII) and (IX) to support matrices suchas agarose, cellulose, dextran, starch, alginate, carrageenan, silica orglass. Again these compounds, and their method of use are well known tothose skilled in the art and, again, since the nub of the presentinvention lies in the nature of the ligand and not in the mode ofattachment, any of these condensing agents will serve in the preparationof the new matrix-ligand conjugates of the invention. As non-limitingexamples of such condensing agents, there may be mentionedN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

As examples of linking units of General Formula (VIII) which may be usedto produce compounds of General Formula (IX) there may be mentioneddiamines such as ethylenediamine, N,N′-dimethylethylenediamine,N-ethylethylenediamine, N-(β-hydroxyethyl)ethylenediamine,propylenediamine, N-methylpropylenediamine,N-(β-hydroxyethyl)propylenediamine, 1,4-diaminobutane,1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane,1,8-diaminooctane, 1,9-diaminononane, 1,10-diaminodecane,1,12-diamindodecane, piperazine, 3-hydroxy-1,5-diaminopentane, m- andp-phenylene diamine, m- and p-aminobenzylamine; amino alcohols such asethanolamine, N-methylethanolamine, N-propylethanolamine,diethanolamine, 3-hydroxypropylamine, 2,3-dihydroxypropylamine,isopropanol amine, 5-aminopentan-1-ol and 6-aminohexan-1-ol;aminophenols such as o-, m- and p-aminophenol, aminocarboxylic acidssuch as glycine, N-methylglycine, 3- and 4-aminobutyric acid,3-aminoisobutyric acid, 5-aminovaleric acid, 6-aminocaproic acid,7-aminoheptanoic acid, m- and p-aminobenzoic acid; aminophosphonic acidssuch as m-aminobenzenephosphonic acid and p-aminobenzylphosphonic acid;and aminoarylene vinylsulphone precursors such asaniline-3-β-sulphatoethylsulphone and aniline-4-β-sulphatoethylsulphone.

The reaction of halogenoheterocyclic compounds of General Formula (IV)with compounds of General Formulae (V), (VI) and (VII) or (VIII) may becarried out in an organic solvent which is not miscible with water; orin an organic solvent which is miscible with water, or in a mixture ofwater and a water miscible organic solvent. Examples of suitable organicsolvents which are not miscible with water are toluene, xylene orchlorobenzene; examples of suitable organic solvents which are misciblewith water are acetone, methyl ethyl ketone or dioxan. The firstreaction of the halogenoheterocyclic compound may be carried out attemperatures between 0° C. and 25° C., ideally between 0° C. and 5° C.;the second reaction may be carried out at temperatures between 20° C.and 50° C., ideally between 30° C. and 45° C. and the third reaction attemperatures between 20° C. and 100° C. During such reactions, theinorganic acid such as hydrochloric acid or hydrofluoric acid which isproduced is neutralised by the use of an acid binding agent such assodium hydroxide, sodium carbonate, sodium bicarbonate, calciumhydroxide or calcium carbonate.

Additionally, compounds of General Formula (IX) may be reacted with areactive polymerisable monomer to form a polymerisable compound ofGeneral Formula (X):

wherein A₁, A₂, B₁, B₂, D₁, D₂, p, X, T, L, V, m, R₁, R₂, R₃, R₄, R₅,R₆, R₇, R₈, and R₉ have the meanings hereinbefore specified; R₁₁represents a hydrogen atom or an alkyl group containing from 1 to 6carbon atoms; R₁₀ represents a carbonyl group, a methylene group, a—NH—CH₂— group or a —S—CH₂— group. Examples of reactive polymerisablemonomers include acryloyl chloride, methacryloyl chloride, allylbromide, allylamine or 3,4-epoxybutene. Polymerisable compounds ofGeneral Formula (X) may be polymerised, optionally in the presence ofother polymerisable monomers, to form affinity ligand matrix conjugatesof General Formula (III). Such polymerisation procedures are well knownto those skilled in the art.

In another embodiment the invention relates to novel affinity ligandmatrix conjugates of General Formula (XI):

wherein A₁, A₂, B₁, B₂, D₁, D₂, p, X, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ have the meanings hereinbefore specified and Halogen represents afluorine, chlorine, bromine or iodine atom.

Furthermore, the invention relates to a method of attaching novelaffinity ligands of General Formula (XI) as defined above to a matrix ofGeneral Formula (VII) as defined above by reacting the novel affinityligands with the matrix at temperatures between 0° C. and 100° C.,optionally in the presence of an acid binding agent.

In another embodiment the invention relates to novel affinity ligands ofGeneral Formula (XII):

wherein A₁, A₂, B₁, B₂, D₁, D₂, p, X, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ have the meanings hereinbefore specified and j is an integerbetween 2 and 20.

Furthermore, the invention relates to a method of preparing above novelaffinity ligands by reacting a compound of above General Formula (XI)with an alkylene diamine of General Formula H₂N—(CH₂)_(j)—NH₂ attemperatures between 0° C. and 100° C., optionally in the presence of anacid binding agent.

In another embodiment the invention relates to novel affinity ligands ofGeneral Formula (XIII):

wherein A₁, A₂, B₁, B₂, D₁, D₂, p, X, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ have the meanings hereinbefore specified; j is an integer between2 and 20, and q is 0 or 1.

Furthermore, the invention relates to a method of attaching novelaffinity ligands of General Formula (XIII) as defined above to a matrixof General Formula (VII) as defined above by reacting the novel affinityligands with the matrix at temperatures between 0° C. and 100° C. in thepresence of a condensing agent. As non-limiting examples of suchcondensing agents, there may be mentionedN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)arbodiimide.

Furthermore, the invention relates to a method of preparing novelaffinity ligands of above General Formula (XIII) by reacting a compoundof above General Formula H₂N—(CH₂)_(j)—(CO)_(q)—OH at temperaturesbetween 0° C. and 100° C., optionally in the presence of an acid bindingagent.

In another embodiment, the invention relates to novel affinity ligandsof above General Formula (X) wherein A₁, A₂, B₁, B₂, D₁, D₂, p, X, T, L,V, m, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ have the meaningshereinbefore specified; R₁₀ represents a hydrogen atom or an alkyl groupcontaining from 1 to 6 carbon atoms; R₁₁ represents a carbonyl group, amethylene group, a —NH—CH₂— group or a —S—CH₂— group; preferably L is analkyl group containing from 4 to 10 carbon atoms, preferably Trepresents a —NH— group, preferably V represents a —NH— group and m ispreferably 1.

In a preferred embodiment, the invention relates to novel affinityligands of General Formula (XIV):

wherein A₁, A₂, B₁, B₂, D₁, D₂, p and R₁ have the meanings specifiedabove.

In another preferred embodiment, the invention relates to novel affinityligands of General Formula (XV):

wherein A₁, A₂, B₁, B₂, D₁, D₂, p and R₁ have the meanings specifiedabove and j is an integer between 2 and 20.

In another preferred embodiment, the invention relates to affinityligands of General Formula (X), (XI), (XII), (XIII), (XIV) and (XV)wherein D1 and D2 both independently represent a guanidino group, animidazole group, a primary amino group, a diethylamino group, atrimethylammonium group or a triethylammonium group.

In another preferred embodiment, the invention relates to affinityligands of General Formula (IX), (X), (XI), (XII), (XIII), (XIV) and(XV) wherein p is 1.

In another preferred embodiment, the invention relates to affinityligands of General Formula (IX), (X), (XI), (XII), (XIII), (XIV) and(XV) wherein B1 and B2 both independently represent a —CHCOOH—(CH₂)₃—group, a —CHCOOH—(CH₂)₄— group, a butyl group, a pentyl group or aphenyl group.

In another preferred embodiment, the invention relates to affinityligands of General Formula (IX), (X), (XI), (XII), (XIII), (XIV) and(XV) wherein A1 and A2 both independently represent a —NH— group.

In another preferred embodiment, the invention relates to affinityligands of General Formula (IX), (X), (XI), (XII) and (XIII) wherein Xrepresents a nitrogen atom.

In another preferred embodiment, the invention relates to affinityligands of General Formula (XII), (XIII) and (XV) wherein j is between 4and 10.

Preferred affinity ligands according to the invention are:

A valuable group of affinity ligand-matrix conjugates is represented bythe General Formula (XXVII):

wherein B₁, B₂, D₁, D₂, p, M, R₁, R₂ and R₃ have the meaningshereinbefore specified and j is an integer between 4 and 10.

An especially valuable group of affinity ligand support matrices isrepresented by the General Formula (XXVIII):

wherein B₁, B₂, D₁, D₂, p, X, M, R₁, R₂, R₃ and M have the meaningshereinbefore specified.

Typically, reaction of compounds of General Formula (XXIX):

with 3-propoxy-(1,2-epoxy) derivatised matrices at temperatures between10° C. and 30° C. in the presence of an acid binding agent producesnovel affinity-ligand matrix conjugates which are of outstanding valuein the binding of endotoxin from water, aqueous solutions, proteins,drugs, blood and plasma.

Preferred affinity ligand matrix conjugates according to the inventionare

wherein M is as defined above.

The invention further covers the use of all such affinity ligand-supportmatrices in the separation, isolation, purification, quantification,identification and characterisation of endotoxin or analogues,fragments, derivatives thereof and precursors.

Endotoxins are a family of lipopolysaccharides, often abbreviated asLPS, which share a common structure. Endotoxins exist in a number offorms, for example the most significant endotoxin types comprising lipidA attached to a core polysaccharide component which may also be linkedto a O-specific chain polysaccharide. The core polysaccharide componentmay consist of an inner core, an inner core attached to an outeroligosaccharide or an inner core attached to an outer core. Endotoxin isknown to be extremely hererogeneous, particularly with respect to theO-Specific Chain polysaccharide and the outer core polysaccharide. Sinceendotoxin is known to be heterogeneous, the term “endotoxin” as usedherein includes all naturally occurring forms which comprise lipid Acovalently linked to a polysaccharide, including analogues, derivatives,fragments and precursors thereof and all such forms, irrespective oftheir source, are subject to the claims of this invention.

Furthermore, the invention relates to a method of attaching the novelaffinity ligands of General Formulae (IX) as defined above, (X) asdefined above, (XII) as defined above, (XV) as defined above and (XXIX)as defined above to carbohydrate or organic polymer matrices by reactingthe carbohydrate or organic polymer matrix with an activating agentfollowed by reaction of the activated matrix with the novel affinityligand, optionally in the presence of an acid binding agent. Theinvention also relates to a method of attaching the novel affinityligands of General Formulae (XIII) as defined above to carbohydrate ororganic polymer matrices by condensation with the matrix. The inventionfurthermore relates to a method of attaching the novel affinity ligandsof General Formulae (IX) as defined above, (X) as defined above, (XII)as defined above, (XV) as defined above and (XXIX) as defined above tometal oxide, glass or silica matrices, optionally coated with an organicpolymer by reacting the optionally coated metal oxide, glass or silicamatrix with an activating agent followed by reaction of the activatedmatrix with the novel affinity ligand, optionally in the presence of anacid binding agent. Another embodiment of the invention relates to amethod of attaching the novel affinity ligands of General Formulae(XIII) as defined above to metal oxide, glass or silica matricesoptionally coated with an organic polymer by condensation with thematrix. In another embodiment the invention relates to a method ofattaching novel affinity ligands of General Formula (XI) as definedabove, (XIV) as defined above and (XVI-XXVI) as defined above to amatrix of General Formula (VII) as defined above by reacting the novelaffinity ligands with the matrix at temperatures between −0° C. and 100°C., optionally in the presence of an acid-binding agent. The inventionalso relates to all the affinity ligand-matrix conjugates, prepared asdescribed in the above methods.

In another embodiment, the invention relates to the use of the affinityligands according to the invention and the affinity ligand-matrixconjugates according to the invention for the separation, isolation,purification, characterisation, identification or quantification ofendotoxin. In another embodiment the invention relates to any processwhereby endotoxin containing solutions or liquids are applied toaffinity ligand-matrix conjugates according to the invention at a pH inthe range 1.0 to 13.0. The invention also relates to a process for theisolation of endotoxin from various fluids such as water, aqueoussolutions, body fluids, blood, plasma, solutions of pharmaceuticalproducts, proteins and other compounds of biological origin by carryingout affinity chromatography using as the biospecific ligand a ligand ofGeneral Formula (I) as defined above.

Another embodiment of the invention relates to the use of affinityligands according to the invention and affinity ligand-matrix conjugatescomprising such ligands according to the invention for theextracorporeal removal of endotoxin from whole blood or plasma which istaken from a donor and re-infused back into the same donor or anotherrecipient following treatment.

The invention will now be described in further detail with reference tothe following Examples. The examples are provided for illustrativepurposes and are not to be construed as limiting the scope of theinvention in any way.

EXAMPLE 1

This Example illustrates the synthesis of a typical affinity ligand ofGeneral Formula (XIV) defined by the reaction of a halogenohetercycliccompound of General Formula (IV) with a compound of General Formula (V)and (VI).

A solution of 1 part cyanuric chloride in 10 parts acetone was addeddropwise to a stirred solution comprising 2 parts L-arginine in 100parts water. The mixture was stirred for 2 hours at 0-5° C. whereuponthe solution was warmed to 30° C. and mixing continued for a further 16hours. The pH was maintained within the range 5.0-7.0 throughout bytitration with 1M sodium hydroxide solution. The reaction product wasprecipitated by the addition of solid sodium chloride to a finalconcentration of 20% (w/v), filtered and dried in-vacuo. TLC analysis(THF/propan-2-ol/water (1:2:1 by vol.) solvent) revealed the presence ofa single reaction product (R_(f) 0.42). The molecular mass of theisolated compound was determined by mass spectroscopy and found to beconsistent with a compound comprising cyanuric chloride derivatised with2 molecules of arginine (calculated M_(r)=459.5; molecular ion (+veFAB)=460.3, (−ve FAB)=458.4). The ¹H-NMR spectrum was consistent with acompound containing arginine.

EXAMPLE 2

This Example illustrates the synthesis of an optionally derivatisedsupport matrix of General Formula (VI).

A solution of 1 part 1,6-diaminohexane in 12 parts water was added to astirred suspension comprising 29 parts epoxy-activated agarose beads (30μmol epoxide groups per g agarose gel) in 48 parts water and stirred for24 h at 30° C. The amino-hexyl agarose gel was filtered and washedconsecutively with 12×29 parts water and allowed to drain under gravityon completion of the final wash. Analysis of the resulting amino-hexylagarose for the presence of primary amines (TNBS asay) and epoxidegroups (thiosulphate/sodium hydroxide titration) revealed completereaction of the epoxide groups with 1,6-diaminohexane.

EXAMPLE 3

Example 2 was repeated by replacing 1,6-diaminohexane with1,4-diaminobutane, 1,5-diaminopentane, 1,7-diaminoheptane,1,8-diaminooctane, 1,9-diaminononane and 1,10-diaminodecane. In allcases amino-alkyl derivatised agarose matrices were obtained.

EXAMPLE 4

This Example illustrates the reaction of an optionally derivatisedsupport matrix of General Formula (VII) with a halogenoheterocycliccompound of general formula (IV).

A solution of 1 part cyanuric chloride in 10 parts acetone was added toa pre-cooled (0-4° C.) suspension of 40 parts amino-hexyl agarose matrixprepared according to Example 2 in 40 parts of 0.5M potassium phosphatebuffer, pH 7.0. The mixture was stirred for 1 hour at 0-4° C., filteredand washed consecutively with 5×40 parts of a solution comprising 1 partacetone and 1 part water, 5×40 parts water, 5×40 parts of a solutioncomprising 1 part acetone and 1 part water and 10×40 parts water.Analysis of the resulting dichlorotriazine-activated agarose matrix forthe presence of amines (TNBS assay) and release of chloride ionsfollowing treatment with 1M sodium hydroxide revealed complete reactionof cyanuric chloride with the primary amino groups on the aminohexylagarose matrix.

EXAMPLE 5

Example 4 was repeated by replacing the product prepared according toExample 2 with the products prepared according to Example 3. Analysis ofthe resulting dichlorotriazine-activated agarose matrices for thepresence of amines (TNBS assay) and release of chloride ions followingtreatment with 1M sodium hydroxide revealed in all cases the completereaction of cyanuric chloride with the primary amino groups on theaminoalkyl agarose matrix.

EXAMPLE 6

This Example illustrates the reaction of the product prepared accordingto Example 4 with a compound of General Formula (VI) and a compound ofGeneral Formula (V) to produce affinity ligand-matrix conjugates ofGeneral Formula (II). All solutions were prepared with pyrogen freewater.

One part arginine was added to a suspension containing 35 parts of theproduct prepared according to Example 4 in 105 parts of 0.1 M sodiumcarbonate buffer, pH 10.25. The mixture was stirred for 24 hours at 30°C., filtered and washed consecutively with 12×35 parts of 0.1 M sodiumcarbonate-buffer, pH 10.25 and allowed to drain under gravity. Thismaterial was re-slurried in 105 parts of 0.1 M sodium carbonate buffer,pH 10.25.

One part N-ε-t-BOC-L-lysine was added to the agarose slurry and themixture agitated for 72 hours at 85° C. The suspension was filtered andwashed consecutively with 12×35 parts of water and allowed to drainunder gravity. The mixture was resuspended in 35 parts 0.1Mtrifluoroacetic acid, stirred for 1 hour at 20° C., filtered and washedconsecutively with 3×35 parts methanol, 12×35 parts of water and allowedto drain under gravity. This procedure is required to remove the t-BOCprotecting group.

EXAMPLES 7 to 14

Table 1 gives further examples of the synthesis of novel affinityligand-matrix conjugates of the invention which were prepared by theabove method but with arginine replaced by the amine compound listed inColumn II of Table 1, and N-ε-t-BOC-L-lysine replaced by the aminecompound listed in Column III of Table 1. The number of the Example isgiven in Column I of Table 1. TABLE 1 I II III  7 Arginine Arginine  8Arginine Ammonia  9 N-im-Trityl-L-histidine Arginine 10N-ε-t-BOC-L-lysine N-im-Trityl-L-histidine 11 N-ε-t-BOC-L-lysineN-ε-t-BOC-L-lysine 12 N-im-Trityl-L-histidine N-im-Trityl-L-histidine 13N-im-Trityl-L-histidine Ammonia 14 N-ε-t-BOC-L-lysine Ammonia

EXAMPLE 15

This Example illustrates the ability of affinity ligand-matrixconjugates of General Formula (III) to bind endotoxin from water.

Affinity ligand-matrix conjugate (150 μl) prepared according to Example6 was added to water (1.5 ml) containing Escherichia coli #055:B5endotoxin (1.5×10⁴ EU) and agitated for 1 hour at 20° C. The sample wascentrifuged and the supernatant assayed for the presence of endotoxin bythe Limulus Amoebocyte Lysate Chromogenic Test. Only 0.1 EU/ml(equivalent to 10 pg/ml) was detected in the supernatant indicatinggreater than 99.99% removal of endotoxin from water.

EXAMPLES 16 to 23

Table 2 gives further examples of the ability of novel affinityligand-matrix conjugates of the invention to bind endotoxin. Theprocedure described above was performed except that the affinityligand-matrix conjugate was synthesised according to the Example numbergiven in Column II of Table 2, the amount of endotoxin remaining in thesupernatant (EU/ml) is given in Column III of Table 2 and the amount ofendotoxin adsorbed (%) by the affinity ligand-matrix conjugate is givenin Column IV of Table 2. The number of the Example is given in Column Iof Table 2. TABLE 2 I II III IV 16 7 1.2 99.99 17 8 1.1 99.99 18 9 1.199.99 19 10 7.3 99.93 20 11 0.4 >99.99 21 12 0.1 >99.99 22 13 1.4 99.9923 14 1.0 99.99

EXAMPLE 24

This Example illustrates the ability of affinity ligand-matrixconjugates of General Formula (III) to isolate endotoxin from proteincontaining solutions contaminated with endotoxin.

Affinity ligand-matrix conjugate (150 μl) prepared according to Example6 was added to water (1.5 ml) containing Escherichia coli #055:B5endotoxin (1.5×10⁴ EU) and human serum albumin (15 mg) and agitated for1 hour at 20° C. The sample was centrifuged and the supernatant assayedfor the presence of endotoxin by the Limulus Amoebocyte LysateChromogenic Test which had been calibrated to detect endotoxin in thepresence of 10 mg/ml human serum albumin. Endotoxin at a concentrationof 50 EU/ml was detected in the supernatant indicating 99.5% removal ofendotoxin from a solution containing 10 mg/ml human serum albumin.

EXAMPLES 25 to 32

Table 3 gives further examples of the ability of novel affinityligand-matrix conjugates of the invention to isolate endotoxin fromprotein containing solutions contaminated with endotoxin. The proceduredescribed above was performed except that the affinity ligand-matrixconjugate was synthesised according to the Example number given inColumn II of Table 3, the amount of endotoxin remaining in thesupernatant (EU/ml) is given in Column III of Table 3 and the amount ofendotoxin adsorbed (%) by the affinity ligand-matrix conjugate is givenin Column IV of Table 3. The number of the Example is given in Column Iof Table 3. TABLE 3 I II III IV 25 7 790 92.1 26 8 169 98.3 27 9 12798.7 28 10 81 99.2 29 11 90 99.1 30 12 277 97.2 31 13 54 99.5 32 14 6699.3

EXAMPLE 33

This Example illustrates the capacity of novel affinity ligand-matrixconjugates of General Formula (III) to bind endotoxin in the presence ofprotein. Affinity ligand-matrix conjugate (150 μl) prepared according toExample 6 was added to water (1.5 ml) containing Escherichia coli#055:B5 endotoxin (7.5×10³ EU to 1.5×10⁵ EU) and human serum albumin (15mg) and agitated for 1 hour at 20° C. The samples were centrifuged andthe supernatants assayed for the presence of endotoxin by the LimulusAmoebocyte Lysate Chromogenic Test which had been calibrated to detectendotoxin in the presence of 10 mg/ml human serum albumin. The totalamount of endotoxin present in the uptake mixture and the amount ofendotoxin adsorbed is given in Table 4. TABLE 4 Total Endotoxin (EU)Endotoxin Bound (%) 7.5 × 10³ >99.9 1.5 × 10⁴ 99.5   3 × 10⁴ 99.7 4.5 ×10⁴ 99.6 7.5 × 10⁴ 99.6 1.5 × 10⁵ 99.3These results demonstrate 1 g of novel affinity ligand-matrix conjugateof General Formula (III) is able to bind 1×10⁶ EU (100 μg endotoxin) inthe presence of 10 mg/ml human serum albumin with an extractionefficiency of greater than 99%.

EXAMPLE 34

This Example illustrates the ability of novel affinity ligand-matrixconjugates of General Formula (III) to bind endotoxin in the presence ofprotein and buffer of varying ionic strength. Affinity ligand-matrixconjugate (150 μl) prepared according to Example 6 was added to water(1.5 ml) containing Escherichia coli #055:B5 endotoxin (7.5×10⁴ EU),human serum albumin (15 mg) and PBS buffer (0 to 200 mM) and agitatedfor 1 hour at 20° C. The samples were centrifuged and the supernatantsassayed for the presence of endotoxin by the Limulus Amoebocyte LysateChromogenic Test which had been calibrated to detect endotoxin in thepresence of 10 mg/ml human serum albumin. More than 99% of the endotoxinpresent was adsorbed for all concentrations of PBS buffer investigated.These results demonstrate that novel affinity ligand-matrix conjugatesof General Formula (III) are able to bind endotoxin with high efficiencyand independently of ionic strength or the presence of protein.

1. An affinity ligand-matrix conjugate comprising the matrix and,conjugated thereto by the group Z, a ligand having the general formula(I):

wherein one X is N and the other X is N, CCl or CCN; A₁ and A₂ are eachindependently O, S or N—R₁ and R₁ is H, C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl,benzyl or β-phenylethyl; B₁ and B₂ are each independently an optionallysubstituted hydrocarbon linkage containing from 1 to 10 carbon atoms; D₁is H or a primary amino, secondary amino, tertiary amino, quaternaryammonium, imidazole, guanidine or amidino group; and D₂ is a secondaryamino, tertiary amino, quaternary ammonium, imidazole, guanidine oramidino group; or B₂-D₂ is —CHCOOH—(CH₂)₃₋₄—NH₂; and p is 0 or 1; withthe proviso that, when each X is N, A₁ is NR₁, A₂ is NH, B₂ is phenyl,D₁ is H, D₂ is amidino and p is zero, then R₁ is not methyl,β-phenylethyl or β-hydroxyethyl.
 2. The conjugate according to claim 1,wherein A₁ and A₂ are each independently N—R₁ wherein R₁ is H, C₁₋₆alkyl, C₁₋₆ hydroxyalkyl, benzyl or β-phenylethyl.
 3. The conjugateaccording to claim 2, wherein A₁ and A₂ are each NH.
 4. The conjugateaccording to claim 1, wherein B₁ and B₂ are each independently—CHCOOH—(CH₂)₁₋₄— or a divalent ethyl, propyl, 2-hydroxypropyl, butyl,pentyl, hexyl, phenyl, naphthyl or cyclohexyl group.
 5. The conjugateaccording to claim 4, wherein B₁ and B₂ are each independently—CHCOOH—(CH₂)₃₋₄— or a divalent butyl, pentyl or phenyl group.
 6. Theconjugate according to claim 4, wherein B₁ and B₂ are each independently—CHCOOH—(CH₂)₃₋₄— and p is
 1. 7. The conjugate according to claim 1,wherein D₁ is H, amino, imidazolyl, guanidine, aminidino,trimethylammonium, triethylammonium, dimethylamino, diethylamino,methylamino or ethylamino.
 8. The conjugate according to claim 1,wherein D₂ is imidazolyl, guanidine, aminidino, trimethylammonium,triethylammonium, dimethylamino, diethylamino, methylamino orethylamino.
 9. The conjugate according to claim 1, wherein p is
 1. 10.The conjugate according to claim 1, wherein each X is N.
 11. Theconjugate according to claim 1, wherein Z is-T-[L-V]_(m)-   (II) wherein T is O, S or NR₂ and R₂ is H or C₁₋₆ alkyl;V is O, S, —COO—, CONH, NHCO, —PO₃H—, NH-arylene-SO₂—CH₂—CH₂— or N—R₃and R₃ is H or C₁₋₆ alkyl; L is an optionally substituted hydrocarbonlinkage containing from 2 to 20 carbon atoms; and m is 0 or
 1. 12. Theconjugate according to claim 11, wherein R₂ and R₃ are each H.
 13. Theconjugate according to claim 11, wherein T is O or NH.
 14. The conjugateaccording to claim 13, wherein T is NH.
 15. The conjugate according toclaim 1, wherein m is 1 and L is a divalent butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl or dodecyl group.
 16. The conjugateaccording to claim 11, wherein m is 1 and V is O, —CONH—, —NHCO— orN—R₃.
 17. The conjugate according to claim 16, wherein V is O or NH. 18.The conjugate according to claim 17, wherein V is NH.
 19. The conjugateaccording to claim 17, wherein Z-M (M being the matrix) is—NH—(CH₂)₄₋₁₀—NH—M.
 20. The conjugate according to claim 19, selectedfrom any of formulae XXX to XXXX.
 21. The conjugate according to claim1, wherein the matrix is optionally tresyl-activated, sulphonylchloride-activated, tosyl-activated, vinyl sulphone-activated orepoxy-activated.
 22. An affinity ligand of general formula (I):

wherein one X is N and the other X is N, CCl or CCN; A₁ and A₂ are eachindependently O, S or N—R, and R₁ is H, C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl,benzyl or β-phenylethyl; B₁ and B₂ are each independently an optionallysubstituted hydrocarbon linkage containing from 1 to 10 carbon atoms; D₁is H or a primary amino, secondary amino, tertiary amino, quaternaryammonium, imidazole, guanidine or amidino group; and D₂ is a secondaryamino, tertiary amino, quaternary ammonium, imidazole, guanidine oramidino group; or B₂-D₂ is —CHCOOH—(CH₂)₃₋₄—NH₂; and p is 0 or 1; andwherein Z is a functional group capable of reaction with a solid matrix;and with the proviso that, when each X is N, A₁ is NR₁, A₂ is NH, B₂ isphenyl, D₁ is H, D₂ is amidino and p is zero, then R₁ is not methyl,β-phenylethyl or β-hydroxyethyl.
 23. The ligand according to claim 22,wherein the matrix is optionally tresyl-activated, sulphonylchloride-activated, tosyl-activated, vinyl sulphone-activated orepoxy-activated.
 24. The ligand according to claim 22, wherein Z is F,Cl, Br or I.
 25. The ligand according to claim 22, wherein Z is—NH—(CH₂)₂₋₂₀—NH₂.
 26. The ligand according to claim 22, wherein Z is—NH—(CH₂)₂₋₂₀—(CO)₀₋₁—OH.
 27. The ligand according to claim 22, whereinZ is-T-[L-V]_(m)-R₁₀—CR₁₁═CH₂ wherein T is O, S or NR₂ and R₂ is H or C₁₋₆alkyl; V is O, S, —COO—, CONH, NHCO, —PO₃H—, NH-arylene-SO₂—CH₂—CH₂— orN—R₃ and R₃ is H or C₁₋₆ alkyl; L is an optionally substitutedhydrocarbon linkage containing from 2 to 20 carbon atoms; m is 0 or 1;R₁₀ is CO, CH₂, NH—CH₂— or —S—CH₂—; and R₁₁ is H or C₁₋₆ alkyl.
 28. Theligand according to claim 24, wherein Z is Cl, and each X is N.
 29. Theligand according to claim 28, wherein each X is N.
 30. The ligandaccording to claim 22, selected from any of formulae XVI to XXVI.
 31. Amethod for the separation, isolation, purification, characterization,identification or quantification of an endotoxin; wherein said methodutilizes an affinity ligand or conjugate having the general formula (I):

wherein one X is N and the other X is N, CCl or CCN; Z is a functionalgroup capable of reaction with a solid matrix; A₁ and A₂ are eachindependently O, S or N—R₁ and R₁ is H, C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl,benzyl or β-phenylethyl; B₁ and B₂ are each independently an optionallysubstituted hydrocarbon linkage containing from 1 to 10 carbon atoms; D₁is H or a primary amino, secondary amino, tertiary amino, quaternaryammonium, imidazole, guanidine or amidino group; and D₂ is a secondaryamino, tertiary amino, quaternary ammonium, imidazole, guanidine oramidino group; or B₂-D₂ is —CHCOOH—(CH₂)₃₋₄—NH₂; and p is 0 or
 1. 32.The method according to claim 31, for the removal of an endotoxin fromwater or an aqueous solution, body fluid, blood, plasma, solution ofpharmaceutical products, protein or other compound of biological origin.33. The method according to claim 32, which comprises using theconjugate for the extracorporeal removal of endotoxin from whole bloodor plasma, prior to re-infusion into the door or another recipient. 34.The method according to claim 31, wherein the endotoxin originates froma Gram-negative bacterium.
 35. The method according to claim 31, in anendotoxin-containing solution or liquid is applied to the conjugate at apH of 1.0 to 13.0.